Download Isolation Purification and Characterisation Isolation, Purification and

Isolation Purification and Characterisation
Isolation,
of Proteins from Plants
Seema Mishra, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2012
In vivo Studyy
Cellular localization of protein
Activity e.g. detection of metal
transport by specific fluorescent dyes
or p
patch clamping
p g
Limitations :
No structural determination
Kobae et al., 2004, PlantCell
Physiol 45, 1749-58
No analysis of binding constant
No analysis of catalytic mechanism
Leitenmaier B, Küpper H (2011)
Plant Cell & Environment 34, 208219
I) Protein Isolation: Sources (expression) of plant proteins
Native expression (i
(i.e.
e in intact organs or in tissue culture)
 Advantages:
- Correct folding
- Correct
C
t post-translational
tt
l ti
l processing
i ((e.g. iinsertion
ti off active
ti centre,
t phosphorylation,
h
h l ti
glycosylation, cleavage, ...)
- No cloning needed
 Disadvantages
- Usuallyy lower yyield
- Often many similar proteins in the same organisms  difficulties with purification
root freezing in liquid nitrogen and hydroponic plant cultivation in Küpper lab
I) Protein Isolation: Sources (expression) of plant proteins
Heterologous expression (e.g. in bacteria, yeast or insect cells)
 Advantages:
- Usually higher yield
- Specific expression of one protein in large amounts and possibly with specific tag
(e g His-tag)
(e.g.
His tag) facilitates purification
 Disadvantages
- Often problems with folding, in particular in the case of large proteins and integral
membrane proteins
p
cofactors ((e.g.
g iron-sulfur clusters)) is difficult or impossible,
p
- Insertion of complicated
other post-translational modifications may be missing or different compared to the
native protein  possibly wrong conclusions about functions/mechanisms
I) Protein Isolation: Methods for isolation
Grinding in liquid nitrogen
 Used for hard tissues and cells like roots, stems, but also for hard-walled cells like
some algae and cyanobacteria
 Low
L
ttemperature
t
protects
t t the
th proteins
t i during
d i grinding
i di
 Time consuming (manual grinding) or requiring suitable machinery (expensive
solution: lab mill, several thousand €, cheap solution: wheat mill, about 200 €)
Liquid nitrogen cooled
wheat mill in Küpper lab
I) Protein Isolation: Methods for isolation
Ultrasound homogenisation
 Used for soft tissues like some leaves, and a post-treatment after grinding
 Does not require freezing and thus may avoid artefacts of freezing, but may cause
artefacts
t f t by
b heating
h ti off the
th sample
l
 Requires expensive (usually several thousand €) machinery
I) Protein Isolation: Methods for isolation
French Press
 Used for individual cells (plant cell culture, algae or bacteria) without or with soft walls
 Does not require freezing and thus may avoid artefacts of freezing
 Requires
R
i
very expensive
i ((usually
ll many th
thousand
d €) machinery
hi
From: www.diversified-equipment.com
From: www.pegasusscientific.com
I) Protein Isolation: Methods for isolation
Lysis buffer
 Used only for bacteria or animal cells
 May cause degradation
 No
N machinery
hi
needed
d d
From: bio-ggs.blogspot.com/2009/11/ggs-live-western...
II) Protein Purification: Overview of Principles
Separation by expression site
 Selective use of tissues or organelles
 Separation of soluble from membrane proteins by centrifugation
Separation by size
 Ultrafiltration
Ult filt ti
 Size exclusion chromatography
 Preparative native gel electrophoresis
Separation by charge
g chromatography
g p y
 Ion exchange
 Isoelectric focussing (as chromatography, in solution or in gel electrophoresis)
Separation by specific binding sites
 Metal affinity chromatography using natural or artificial metal binding sites
 Immuno-Chromatography
Immuno Chromatography using immobilised antibodies
 Magnetic separation using magnetically tagged antibodies
II) Protein Purification: Separation by size in native gels
 Principle: Small proteins are less retained by the fibers of the gel than large proteins,
so that small proteins migrate faster
foto of a green gel (Küpper et al., 2003, Funct Plant Biol)
From: www.columbia.edu/.../c2005/lectures/lec6_09.html
III) Protein Identification: Overview of Principles
Size determination
 Size exclusion chromatography or SDS PAGE
 Comparison with expected size of protein (known e
e.g.
g from reference or cDNA)
Western
W
t
Blotting
Bl tti
 Binding to specific primary antibody, detected via labelled or enzymatically active
secondary antibody
Biochemical assays in native gels
de t cat o of
o enzymes
e y es by their
t e characteristic
c a acte st c act
activity
ty
 Identification
 Identification of metalloproteins by their metal content
Mass Spectrometry
 Fragmentation of the protein, identification of fragment sizes, and subsequent
comparison
i
tto a library
lib
off kknown ffragmentation
t ti patterns
tt
N-terminal Seqencing (Edman degradation)
 Sequential chemical removal of individual amino acids from the N-terminus
III) Protein Identification by assays in native gels
 Example
p of a metalloprotein
p
identification in native g
gels: Scanning
g of the g
gels with
laser ablation inductively coupled mass spectrometry (LA-ICP-MS) for obtaining
information about metal binding to individual bands of a gel
from: Polatajko A, Azzolini M, et al (2007) JAnalAtomSpect 22, 878-887
III) Protein Identification by assays in native gels
 Example of a biochemical assay in native gels: identification of Cd-Carboanhydrase
by its metal content combined with an in-gel carboanhydrase assay
f
from:
Lane
L
TW,
TW Morel
M l FMM (2000) PNAS97,
PNAS97 4627
4627-4631
4631
III) Protein Identification: Mass Spectrometry
Principle
1) protein band is cut of out gel
2) protein is digested into peptides
3) mass spectrometry
4) comparison of the fragment sizes with a database
5) assignment of likely sequences to fragments
6) comparison of the fragment sequences with a database
7) result: list of proteins from the database that have a similar fragmentation pattern
from: Gingras AC et al
al, 2004
2004, J Physiol 563
563, 11
11-21
21
III) Protein Identification: Mass Spectrometry
3) mass spectrometry
4) comparison of the fragment sizes with a database
from: http://www.med.monash.edu.au/biochem/facilities/proteomics/maldi.html
http://www med monash edu au/biochem/facilities/proteomics/maldi html
III) Protein Identification: Mass Spectrometry
3a) further fragmentation of one of the fragments from the digest in the mass spectrometer, then
again mass spectrometry (MSMS)
p
of the fragment
g
sizes with a database
4a)) comparison
from: http://www.med.monash.edu.au/biochem/facilities/proteomics/maldi.html
III) Protein Identification: Mass Spectrometry
 5) assignment of likely sequences to fragments
 6) comparison of the fragment sequences with a database
 7) result: list of proteins from the database that have a similar
fragmentation pattern
from: http://www.med.monash.edu.au/biochem/facilities/proteomics/maldi.html
III) Protein Identification: Mass Spectrometry
from: Gingras AC et al, 2004, J Physiol 563, 11-21
Limitations and artefacts
 contamination of gel bands with other proteins  identification of the contamination
(very common: keratin from skin!) instead of the protein of interest
 not all proteins have the same detection efficiency (problems e.g. if many cysteines
present))  even small contaminations sometimes lead to wrong
p
g identifications
 not all proteins are in the databases  database may show results that have a similar
fragmentation pattern, but are otherwise unrelated
III) Protein Identification: N-terminal sequencing
Principle
identification of the
amino acid by
y
reversed phase HPLC
and comparison with
HPLC of standard
mixture
from: www.ufrgs.br/depbiot/blaber/section4/section4.htm
III) Protein Identification: N-terminal sequencing
Sample preparation
 1) run acrylamide gel (denaturating or native)
 2) blot onto PVDF (NOT nitrocellulose) membrane using glycine
glycine-free
free buffer
 3) stain the membrane with ponceau red (CBB and other stains also work)
 4) submit for sequencing
Limitations and artefacts
 problems with contaminations
 in eukaryotic proteins, the N-terminus is often blocked (e.g. by methylation), which
equ ed co
complicated
p cated de
de-blocking
b oc g p
procedures.
ocedu es Also
so non-polymerised
o po y e sed ac
acrylamide
y a de
required
remains in the gel can cause blocking, so let the gel polymerise over night
 Cys residues cannot be detected, and also glycosylated redidues may appear as
blank spaces in the sequence
IV) Protein Characterisation: Overview of Principles
Size determination
Charge determination
Analysis of cofactors
Analysis of the 3-dimensional Structure
Activity tests
IV) Protein Characterisation: Size determination
Comparison with predicted size by native and denaturating gel electrophoresis and by
size exclusion chromatography can show native oligomerisation, post-translational
modification but also artefactual degradation/aggregation
200 KDa
116 KDa
97 KDa
66KDa
45KDa
31 KDa
SDS gel and
Western blot of
TcHMA4
 size shows
post-translational
p
processing as
cDNA sequence
predicts 128kDa
(Parameswaran,
Leitenmaier et al.,
2007, BBRC)
IV) Protein Characterisation: Charge determination
 Isoelectric focussing (IEF) can reveal the isoelectric point where the net charge of the
protein is zero, i.e. balance between protonation of carboxyl groups and deprotonation
of amino groups is achieved
pH
alkaline region
neutral
t l region
i
acidic region
from: commons.wikimedia.org
IV) Protein Characterisation: Analysis of cofactors
Identification and reactivity: UV/VIS Absorption and fluorescence Spectroscopy
 Metal content (about 30% of all proteins are metalloproteins!): AAS, ICP-MS/OES,
EDX/PIXE/XRF
UV/VIS spectrum
of TcHMA4
 Cadmium
g causes
binding
ligand-metalcharge-transfer
(LMCT) bands
indicating
cysteine ligation
(Leitenmaier and
Küpper unpublished)
Küpper,
IV) Protein Characterisation: Analysis of cofactors
Metal ligands: EXAFS, EPR, heteronuclear NMR provide information about ligand types
and their spatial arrangement around the active site (each of these techniques has
different strenghts)
EXAFS spectrum
of Zn-TcHMA4
 mixed S/N
g
, multiple
p
ligands,
scattering shows
histidine
contribution
(Leitenmaier and
Küpper, unpublished)
IV) Protein Characterisation: Analysis of the 3D Structure
Circular Dichroism (CD) Spectroscopy
From: www.proteinchemist.com/cd/cdspec.html
 information about proportions of secondary structure types in a protein
 particularly useful when X-ray crystallography and NMR are not applicable
IV) Protein Characterisation: Analysis of the 3D Structure
X-ray crystallography
E
Example:
l iin th
the nickel-binding
i k l bi di transcription
t
i ti factor
f t NikR,
NikR th
the mechanism
h i
was concluded
l d d
from the X-ray structure
From: Schreiter ER et al., 2006, PNAS103, 13676-81
 Ni binding causes RHH domains to rotate about the flexible interdomain linkers to
orient their antiparallel -strands toward the same face of the repressor, allowing each
to occupy the DNA major groove of an operator palindrome half
half-site
site
 Binding of Ni creates a surface of the MBD suitable for interacting with DNA by
stabilization of a helix and a loop
IV) Protein Characterisation: Activity tests
Titration of an enzyme with its substrate(s) reveals binding constants and possible
substrate
b t t inhibition
i hibiti
Activation of a human
Cu-ATPase
Hung et al. Biochem.J.
2007, 401
TcHMA4 Fraction3 051108
350
Both are P1B-Type ATPases
showing the same activation
pattern by
p
y „„their“ metal after
reconstitution into artificial
lipid vesicles
Zn
Z
Cd
300
turnover rate[1/s]
250
200
Activity of TcHMA4
150
(purified by
Leitenmaier and
Kü
Küpper)
)
100
50
0
0,01
0,1
1
conc. metal[µM]
10
IV) Protein Characterisation: Activity tests
Two-dimensional data, e.g. substrate concentration and temperature reveal insights
into interactions between factors that are decisive for enzyme activity, e.g.
temperature dependence of substrate binding and influence of the substrate on the
thermostability of the enzyme
Activity of TcHMA4
(Leitenmaier et al (2011) Biochimica et Biophysica Acta (Biomembranes) 1808, 2591-2599)
Example: Flowchart of expression, isolation, purification and
characterisation of the Cd/Zn-ATPase TcHMA4
Grow plants expressing TcHMA4 hydroponically, harvest and freeze roots
Grind the frozen roots with isolation buffer in liquid
q N2, thaw
Centrifuge,
g discard the supernatant
p
((soluble pproteins)) and resuspend
p
the
pellet (membrane proteins) with solubilisation buffer
Centrifuge for removing insoluble residue, collect solubilised protein
Immobilized Metal Affinity Chromatography on Ni-IDA column
Identify:
Quantify: In SDS
Western-Blotting, gel via fluorescent
f
Edman Sequ.
dye
Metal binding:
AAS/ICP,
S/ C EXAFS,
S
UV/VIS
Activity tests
( catalytical
properties)
The slides can be downloaded from the workgroup
homepage
www.uni-konstanz.de
ni konstan de  Department of Biology
Biolog  Workgroups
Workgro ps  Küpper lab,
lab
or directlyy
http://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html